1. Field of the Invention
The present invention relates to a complex in which lactoferrin holds folic acid and/or vitamin B12 (hereinafter referred to as folic acid and/or vitamin B12-lactoferrin complex), a method for the production thereof, and use thereof. A folic acid and/or vitamin B12-lactoferrin complex of the present invention is characterized in that folic acid in the complex exhibits improved photostability, acid tolerance and solubility as compared to folic acid present by itself, and vitamin B12 in the complex exhibits improved acid tolerance as compared to vitamin B12 present by itself, and accordingly is useful as a material for foods, drinks and medicines.
2. Description of the Related Art
Folic acid which was discovered as an antianemic factor a long time ago is one of the vitamins essential for the body. Further, various enzymes are known to require folic acid as a coenzyme. Also, folic acid is extensively involved in the metabolisms of nucleotides, glycine, histidine, or the like, and the biosyntheses of proteins. Pteroylglutamate, 7,8-dihydropteroylglutamate, or the like and polyglutamin compounds thereof are also known as folic acid in a broad sense, and all of these compounds have physiological activities as folic acid. A deficiency in folic acid is known to cause abnormal myelopoietic functions (megaloblastic anemia), nervous disorder, intestinal dysfunction, or the like.
Recently, the higher probability of newborns having abnormal neural tubes due to a nutrient deficiency in mothers during pregnancy having particularly attracted attention in Europe and the United States, it has been revealed that this risk can be reduced by administering folic acid (Czeizel, A. E., J. Pediat. Gastroenterol. Nutr., vol. 20, pp. 4-16, 1995). Furthermore, it has been reported that an increase in homocysteine, which is associated with heart diseases, and a depletion of folic acid in the serum occur simultaneously (Jacob, R. A., M. M. Wu, S. M. Henning and M. E. Swendseid, J. Nutr., vol. 124, p. 1072, 1994). It has been also reported that folic acid exerts a preventive effect on cancer, particularly epithelial cancer (Glynn, S. A., D. Albanes, Nutr. Cancer, vol. 22, p. 101, 1994). Thus, the importance of folic acid is being recognized again.
On the other hand, folic acid is known to be relatively heat stable in the absence of oxygen. However, folic acid has low heat stability and storage stability in the presence of oxygen. For example, about 60% of folic acid was reported to be lost in cow""s milk by heat sterilization or during storage (Renner E., Japanese Journal of Dairy and Food Science, vol. 35, p. A121-A135, 1996), and 25% of folic acid was lost in powdered milk (Oamen E. E., Hansen, A. P. M. and Swartzel, K. R., J. Dairy Sci., vol. 72, pp. 614-619, 1989). Furthermore, it is also known that folic acid is extremely sensitive to and rapidly decomposes in light and must be handled in the dark (Henderson B. G., Annu. Rev. Nutr., vol. 10, pp. 319-335, 1990).
The presence of a folic acid-binding protein in cow""s milk is conventionally known (Ford, J. E., D. N. Salter and K. J. Scott, J. Dairy Res., vol. 36, p. 435, 1969). This folic acid-binding protein has a molecular weight of about 25 kDa, binds one molecule of folic acid, and is suggested to promote absorption from the intestinal tract (Said H. M., F. K. Ghishan and R. Redha, Am. J. Physiol., vol. 252, p. G229, 1987). However, the content of this folic acid-binding protein in milk is as low as 10 mg/L (Parodi P. W., Diet and Health News for New Zealand Health Professionals, vol. 27, pp. 1-4, 1998). Further, there has been no report on the photostability of this folic acid-binding protein.
As described above, folic acid has lately attracted considerable attention because of its important physiological functions, and the use of folic acid as a material for foods, drinks and medicines is desirable. However, there exists various limitations; for example, it is difficult to use folic acid, particularly in drinks, because of its poor photostability and solubility, which occasionally causes precipitation or requires shielding in packaging. Furthermore, as for the folic acid-binding protein, its content in milk is small and the outlook for its industrial scale production is still in doubt.
On the other hand, since vitamin B12 is associated with the synthesis of heme, which is necessary for erythropoiesis, its deficiency is known to generate megaloblasts and cause anemia. Furthermore, since vitamin B12 is essential for the generation and growth of cells, its deficiency causes inflammation of mucosal tissues, diarrhea, or the like. Vitamin B12 is also known to have important roles in the reproductive functions and nervous system (Toru Fukui, xe2x80x9cHow to Take Vitamins and Mineralsxe2x80x9d, Maruzen, 1997). Vitamin B12 deficiency is attributed mainly to a vegetarian diet or often to gastrocectomy or abnormal absorption from the intestinal tract. Two extrasecretal proteins are known to be associated with vitamin B12 absorption, i.e., haptocorrin secreted from the salivary gland (Toyoshima S., H. Saido, F. Watanabe, K. Miyatake and Y. Nakano, Abstract of XV International Congress of Nutrition, 204, 1993) and the intrinsic factor secreted from the stomach (Levine J. S., P. K. Nakane and R. H. Allen, Gastroenterology 79, 493, 1980); but the absorption mechanism is complicated. The recommended daily intake of vitamin B12 for an adult in Japan is set to a low 2.4 xcexcg. If this vitamin B12 intake is obtained from food products, loss by cooking has to be taken into consideration. Furthermore, the recommended intake of vitamin B12 per day for an adult in the United States, i.e., Optimal Daily Allowance (ODA), is set as 10 to 300 xcexcg. A more positive intake of this vitamin will be required.
The presence of proteins which bind to vitamin B12 in cow""s milk has been suggested (Peter, W. P., Australian J. Dairy Tech., 53, 37-47, 1998). However, the whole picture of vitamin B12 binding proteins has not yet been revealed. Vitamin B12-binding bovine serum albumin (BSA) is the only one known protein (U.S. Pat. No. 4,082,738) and is being used in the quantification of erythrocytes (Japanese Patent Publication S57-5005281).
Further, vitamin B12 is known to be relatively heat stable but not acid tolerant (Owen R. Fennema, Food Chemistry 3nd ed., Dekker, N.Y.). Accordingly, there is a need for a method to improve the stability, in particular the acid tolerance, of vitamin B12 which has useful physiological functions.
Thus, both folic acid and vitamin B12 are effective substances to ameliorate anemia or the like, but problems in stability restricts their use to limited areas.
In the present invention, it was newly found that lactoferrin interacts with folic acid and vitamin B12 to form a folic acid and/or vitamin B12-lactoferrin complex. Moreover, the folic acid in the complex has increased photostability and drastically increased solubility and is even stable in acid, as compared to folic acid present by itself, and vitamin B12 in the complex has increased acid tolerance as compared to vitamin B12 present by itself. Thus, the present invention has been completed. Accordingly, an object of the present invention is to provide a folic acid and/or vitamin B12-lactoferrin complex in which folic acid and/or vitamin B12 are incorporated into lactoferrin and a method for producing the same. Another object of the present invention is to provide medicines or foods and drinks prepared by using the folic acid and/or vitamin B12-lactoferrin complex.
Lactoferrins to be used in the present invention can be those derived from milk, blood or the like. The source materials are not particularly restricted and can be derived from humans, bovines, hogs, or the like. Lactoferrins obtained by gene recombination can also be used. These lactoferrins can be those purified or partially purified, or materials having a low purity, such as WPC and skimmed milk powder. These lactoferrins can be used with or without heat treatment such as sterilization regardless of the state of bound iron. Further, lactoferrins carrying more than 2 iron molecules can be used (Japanese Patent Laid-open, No. H06-239900 and Japanese Patent Laid-open, No. H7-304798). It is also possible to use peptides which can be obtained by hydrolyzing lactoferrins with enzymes, such as pepsin and trypsin, or acid or alkaline. Further, in the present invention, lactoferrins also include transferrin and ovotransferrin. Lactoferrins used in the present invention thus imply the abovementioned lactoferrin-related substances.
First, a folic acid-lactoferrin complex will be explained as follows.
Folic acid to be used in the present invention is not particularly restricted and can be any products of a grade used for medical and pharmaceutical purposes to that used as a food additive.
A folic acid-lactoferrin complex can be prepared by mixing an aqueous folic acid solution and an aqueous lactoferrin solution, or by dissolving a powder of a mixture of folic acid and lactoferrin. Further, an aqueous solution in which sodium hydroxide, sodium bicarbonate, ammonium bicarbonate, sodium carbonate, and the like are mixed can be more effectively used since lactoferrins can incorporate a large amount of folic acid in an alkaline condition. The amount of folic acid being incorporated is maximally about 200 molecules per one molecule of lactoferrin.